The Frequency of Exotoxin A and Exoenzymes S and U Genes Among Clinical Isolates of Pseudomonas aeruginosa in Shiraz, Iran

Pseudomonas aeruginosa as an opportunistic pathogen produces several virulence factors. The most important of these factors are exotoxin A and type III secretion system (T3SS). The aim of this study was to determine the frequency of toxA, exoU and exoS genes among clinical isolates of P. aeruginosa. In this cross-sectional study from September 2011 to February 2012, 156 P. aeruginosa isolates were recovered from different clinical samples. Susceptibility testing against 10 antibiotics was performed on individual isolates by the disc diffusion method according to CLSI guidelines. Extracted DNA was subjected to PCR assay for determining the presence of toxA, exoU and exoS genes. Overall, the frequency of toxA, exoU and exoS genes were 90.4%, 66.7% and 65.4%, respectively. All of the abdominal and eye isolates were exoS+. The frequency of exoS+/exoU- and exoS-/exoU+ genotypes was estimated 19.2% and 16.2%, respectively. Indeed, genotypes exoS+/exoU+ and exoS-/exoU- were found with frequencies of 48.7% and 15.3%, respectively. The highest and lowest antibiotic resistance rate was seen against azteroenam (94.2%) and amikacin (44.9%), respectively. Fluoroqinolone-resistant isolates were isolated with frequency of 45.8%. Multi-drug resistant (MDR) isolates were detected in 62.8% of isolates. The resistance rate in exoU+ isolates was 86% compared to 66% in exoS+ isolates. The high frequencies of virulence genes detected in our clinical isolates with notable antibiotic resistance rates indicate the potential risk of these isolates in nosocomial infections.

Proliferation of P. aeruginosa in host cells and overcoming defense mechanisms is due to the transfer of many proteins via specialized secretion apparatuses including the types I,II,III,V and VI secretary systems (TSS) (2,8).
ExoA is the major member of the type II secretion system (T2SS) which inhibits protein synthesis by ADP-ribosylation of eukaryotic elongation factor 2 (1,8,9). Another important virulence factor recently recognized is the type III secretion system (T3SS) (3,6). T3SS is a contactdependent protein secretion pathway that plays a major role in the pathogenesis of serious P. aeruginosa infections (10). The four well known T3SS effectory molecules are exoenzymes (Exo) S, U, T and Y (2). It is known that these effectors are delivered to the host cells via a translocation complex consisting of products encoded by the pcrGVHpopBD operon that cause cell necrosis and modulation of actin cytoskeleton, allowing the bacteria to invade the eukaryotic cells and escape phagocytosis (3,6,10).
ExoS is a major cytotoxin involved in stages of colonization, invasion and dissemination of infection (11). ExoU is a potent cytotoxin with phospholipase activity, capable of killing a variety of eukaryotic cells in vitro (12,13). Additionally, ExoU has a greater effect than other T3SS effectors on the virulence of the bacteria (10).
A key determinant of P. aeruginosa is its remarkable resistance to antibiotics and notably many of isolates are multidrug-resistant (MDR) (14). In several studies, the relationship between MDR isolates and presence of genes encoding T3SS, especially ExoU has been demonstrated (10,14).
As the presence of T3SS encoding genes in clinical isolates of P. aeruginosa is a variable trait, distribution of these genes in different populations should be explored. Moreover, to the best of the authors' knowledge there is no previous report from Shiraz investigating the prevalence of P. aeruginosa virulence genes. Therefore, the present study aimed to evaluate the frequencies of toxA, exoS and exoU genes among the different clinical isolates of P. aeruginosa. We also sought to determine whether there is any correlation between the presence of these genes and antibiotic resistance profile of the isolates.

Study design and clinical specimens
In this cross-sectional study, a total 156 P.
aeruginosa clinical isolates were obtained from September 2011 to February 2012 in Shiraz (a major city in the south of Iran) teaching hospitals.

Bacterial identification
All the isolates were identified as P. aeruginosa using both the conventional microbiologic (e.g., Gram staining, capacity for growth at 42 °C, oxidase, and IMViC tests) methods and Microgen TM GnA+B-ID System (Microgen Bioproducts Ltd, U.K) diagnostic kit. Confirmed P.

Antibiotic susceptibility testing
The antimicrobial susceptibility test was done by disk diffusion method on Muller-Hinton agar (Merck Co., Germany). The following antibiotics

Genomic DNA purification and molecular assay
Genomic DNA was extracted from overnight TSB cultures of P. aeruginosa isolates using the small-scale phenol-chloroform extraction method (16). The evaluation of toxA, exoS and exoU genes was accomplished by previously described primers (9,17). PCR amplification was performed in 50 µl   102 (65.4) a Frequency of toxA among wound samples was significantly higher than urine isolates (P< 0.05) and distribution of toxA among other sources was not significantly different. b Distribution of exoS and exoU among clinical samples showed no significant differences. c Significant differences of toxA frequency compared to exoS among individuals clinical samples (P< 0.05). d Significant differences of toxA frequency compared to exoU among individuals clinical samples (P< 0.05).

Results
Of the total 156 P. aeruginosa isolates, majority of isolates were recovered from urine (n= 57) and sputum (n= 35) specimens (

Discussion
As detection of virulence genes in clinical isolates of P. aeruginosa is important (3,18), thus, in the present study, the frequency of some P. aeruginosa virulence genes among different clinical isolates was characterized. As among T3SS, exoT and pcrV (part of the injection apparatus of the T3SS) genes exist in nearly all P. aeruginosa isolates from both clinical and environmental origins (3,12,19), the presence of these genes was not evaluated in this study.
In our investigation, 100% of P. aeruginosa isolates from the AD, ET, eye, blood and wound samples were toxA + . The exoS gene was also detected in 100% of isolates from the AD and eye samples. According to our results, the relative frequencies of virulence genes were higher in some special clinical specimens. It has been suggested that the infection site and duration of disease influences the virulence of P. aeruginosa clinical isolates by altering the production of some virulence determinants. For example, some anatomical sites enhance the production of ExoA and ExoS (10).
In a research in Poland by Wolska et al., the prevalence of toxA among the 62 clinical isolates of P. aeruginosa was 88.7%, which is comparable with our study. However, the frequency of exoS was more than that of the present study (75.8% vs. 66.7%) (17). It seems that the exoU had been acquired through a mobile element (plasmid) integrated into the chromosome of P. aeruginosa.
Therefore, the lower prevalence of the gene than the other virulence genes could be due to this phenomenon (11). However, in our analysis, this rate was nearly the same as exoS prevalence (65.4% vs. 66.7%). In the study conducted by Mitove et al. clinical isolates of P. aeruginosa (14). Moreover, in a study conducted on 55 keratitis isolates, 64% of isolates contained exoS +/ exoUgenotype, whereas 33% and 4% were exoS -/exoU + , and exoS + /exoU + , respectively (18).
It is generally suggested that isolates from clinical setting contain either exoS or exoU gene but not both (22 (20). It has been suggested that the clinical isolates comprising exoU gene were significantly associated with MDR phenotype (18).
This finding is supported in our results, since MDR resistance rate in exoU + isolates with 80% frequency was higher compared to 66% in exoS + isolates. The prevalence of such high resistant P. aeruginosa in our region is not uncommon, since, previously Anvarinejad et al. and Sarhangi et al. showed high rate of MDR among isolates from the burn patients and clinical isolates from Shiraz City, respectively (24,25).
One of the limitations of this study was the small sample size of specimens such as blood, CSF, which could affect the results. However, beside the limitations, the current study has some outcomes.
First we showed a relatively high frequency of toxA, exoS and exoU genes among P. aeruginosa clinical isolates obtained in our area and this frequency was associated to high antibiotic resistance among the isolates. Second, it seems that the source of bacterial isolation is associated with the trend of acquisition of specific virulence genes and these genes may serve to cause specific infections. These results indicate the potential risk of these isolates in nosocomial infections which merit more attention. Of course, further studies are required with larger sample size and from other regions of country to reach a comprehensive conclusion.